Immunogenetic and immunological aspects of rheumatoid arthritis : DERAA and anti-citrulline reactivity can make the difference

Full text

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Immunogenetic and Immunological

aspects of

Rheumatoid Arthritis

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Het onderzoek beschreven in dit proefschrift werd uitgevoerd op de afdeling Reumatologie en Immunohematologie en Bloedtransfusie van het Leids Universitair Medisch Centrum (LUMC) te Leiden en werd financieel ondersteund door een VIDI subsidie van dr. R.E.M. Toes verkregen van NWO.

Drukwerk: GVO drukkers & vormgevers B.V. Ponsen & Looijen. ISBN: 978-90-6464-379-8

Cover: The illustration is designed by drs. C. Röst and the lay-out of the cover and title pages is performed by ir. R. Schoemaker.

Lay-out: dr. P.D. Feitsma

Financial support for the publication of this thesis was provided by Novartis Pharma B.V.; Abbott B.V.

© 2010 Anouk L. Feitsma

The articles are used by permission of the indicated publishers.

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Immunogenetic and Immunological

aspects of

Rheumatoid Arthritis

DERAA and anti-citrulline reactivity can

make the difference

PROEFSCHRIFT

ter verkrijging van

de graad van Doctor aan de Universiteit Leiden,

op gezag van Rector Magnificus prof. mr. P.F. van der Heijden, volgens besluit van het College voor Promoties

te verdedigen op donderdag 11 februari 2010 klokke 13.45 uur

door

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Promotiecommissie

Promotores: prof. dr. T.W.J. Huizinga prof. dr. R.R.P. de Vries

Co-promotor: dr. R.E.M. Toes

Overige leden: prof. dr. F. Koning prof. dr. J.J. van Rood dr. A. Ioan-Facsinay

dr. W. van Eeden

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Ieder schepsel, ieder wezen Is als prentenboek te lezen

En houdt ons een spiegel voor: Ons bestaan en ons verscheiden, Onze vreugde en ons lijden

Geeft het ons in tekens door.

Dichter: Alanus Ab Unsulis

Ca. 12e eeuw, Frankrijk

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Contents

Chapter 1

General Introduction 9

Chapter 2

Protective effect of non-inherited maternal HLA-DR antigens 25 on Rheumatoid Arthritis development

PNAS 2007;104(50):19966-19970

Chapter 3

Protection against Rheumatoid Arthritis by HLA: Nature and Nurture 39 Ann Rheum Dis 2008;67:iii61-iii63

Chapter 4

Identification of citrullinated vimentin peptides as T cell epitopes 51 in HLA-DR4 positive RA patients

Arthritis Rheum, in press

Chapter 5

Risk of progression from undifferentiated arthritis to rheumatoid arthritis: 71 the effect of the PTPN22 1858T-allele in anti-citrullinated peptide

antibody positive patients

Rheumatology (Oxford) 2007;46(7):1092-1095

Chapter 6

A single nucleotide polymorphism in CD40 associates with the rate of 81 joint destruction in Rheumatoid Arthritis

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Chapter 7

General discussion 95

Summary 115

Nederlandse samenvatting 121

List of abbreviations used in this thesis 127

Curriculum vitae 129

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Chapter 1

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Undifferentiated and Rheumatoid Arthritis

Arthritis is a group of conditions characterized by inflammation of the joints. This inflammation can lead to breakdown of the cartilage of the joints that can be caused by different mechanisms, e.g. autoimmunity, fractures, wearing, or infection. The different types of arthritis are diagnosed according to disease criteria, leaving cases that cannot be classified in one of the accepted categories of rheumatic diseases (usually referred to as ‘undifferentiated arthritis’ (UA)). The diagnosis of rheumatoid arthritis (RA), an inflammatory autoimmune disorder characterized by a chronic inflammation of the synovial tissue of several joints, is based on a list of seven criteria developed by the American College of Rheumatology (1). These criteria include clinical, radiological and laboratory findings; i.e. morning stiffness, arthritis of three or more joint areas, arthritis of hand joints, symmetric arthritis, serum rheumatoid factor, rheumatoid nodules, and radiographic changes. The RA patient population is clinically hetero-geneous since only four of these seven ACR criteria have to be fulfilled for the diagnosis of RA. The occurrence of RA varies among countries and areas over the world, but has a prevalence of approximately 1% in Europe (2;3). In the Dutch population, women are affected by RA approximately two times more frequently than men (4).

In the Leiden Early Arthritis Clinic (EAC), which provides an inception cohort of patients with recent onset arthritis (5), 37% of the patients are diagnosed with UA and about 20% with RA at their first visit. After 1 year, 32% of the UA patients have qualified for the diagnosis of RA, indicating the complexity of a diagnosis at initial presentation.

RA patients can develop different kind of autoantibodies, amongst others against citrullinated proteins (anti-citrullinated protein antibodies (ACPA)). Citrullination is a post-translational conversion (deimination) of Arginine to Citrulline residues per-formed by the enzyme peptidylarginine deiminase (PAD) (Figure 1) that results in a small change in molecular mass (<1 Da) and the loss of one positive charge. Although citrullination is a common natural process, these ACPA are specific for RA, and can be measured already years before symptomatic disease (6;7). Recently, it has been shown that ACPA+ and ACPA- RA patients show a different disease course, probably indicating that ACPA+ and ACPA- RA reflect a totally different disease (8-10).

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Progression and Severity scoring

RA is characterized by the proliferation of synovium and the destruction of cartilage and bone during the progression of the disease. Destruction of the cartilage is a consequence of pro-inflammatory cytokines and enzymes that are released during the chronic inflammation process inducing enhanced breakdown of cartilage matrix and reduced synthesis of matrix components by the articular chondrocytes (11;12), but joint erosion results more directly from osteoclasts (13). The formation of pannus, which results from the proliferating synovium (14-16), will eventually lead to joint space narrowing and joint erosions. This is, at least in part, mediated by fibroblast-like synoviocytes from the synovium (17).

Radiographic joint damage is an important outcome measure in RA, in addition to assessments of physical function and disease activity, which all associate with each other (18). It reflects cumulative disease activity and is related to overall disability (19;20). Therefore, progression rates are also influenced by the current therapy, i.e. disease modifying anti-rheumatic drugs (DMARDs) (21;22). Several scoring methods for the assessment of radiographic joint damage exist, from which the most well known are the Larsen (23) and Sharp-van der Heijde method (24;25). Both methods score the individual joints of the extremities but the Sharp-van der Heijde method scores hands and feet for the amount and severity of erosions and joint space narrowing separately (25). The Sharp-van der Heijde method is sensitive to detect changes over time and shows reliable results since it has a low measurement error (26). Analyses of radiographic progression can be analyzed on the individual patient or a patient group level. For both applies that the radiological progression is linear in the first

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(approximately) five years but further in time, the curve levels off to a plateau (18;27-29).

Because nowadays, RA patients are seen in an earlier phase of the disease, before the appearance of well established indicators of poor prognosis such as erosions and nodules, markers which have a good predictive value on radiographic damage in an early phase of the disease will become more important.

Genetic Risk Factors for RA

The pathogenesis of RA is, as in many other autoimmune diseases, complex and largely unknown. It is generally accepted that both genetic and environmental factors contribute and probably also interact with each other. It has been described that genetic factors contribute for about 2/3 to the development of RA (30;31). The contributing risk factors can differ for the susceptibility to, and the progression of RA.

The strongest genetic risk factor, both for susceptibility and severity, has been mapped to the HLA-class II region, most probably DRB1. HLA-class II molecules consist of an

- and -chain which both have constant and variable regions. The variable regions constitute the binding groove for the peptide to be presented by HLA molecules to T cells of the immune system (Figure 2). A particular part of the binding groove, the third hypervariable region, is involved in the susceptibility to RA development. At position 70 to 74 in this third hypervariable region, different variants of amino acid sequences are present. Certain HLA-DRB1 alleles share common epitopes at this position. Regarding the risk for RA development, three variants can be discriminated; either amino acids of the so-called shared epitope (SE), the sequence “DERAA”, or ‘neutral’ amino acids are present. Compared to the ‘neutral’ HLA-DRB1 alleles, carriership of HLA-DRB1 alleles with the SE increases the risk for RA development, and “DERAA”-containing HLA-DRB1 alleles decrease the risk. Both the SE and the “DERAA”-containing HLA-DRB1 allele effects will be discussed in more detail below.

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Shared Epitope and ACPA

HLA-DRB1 molecules containing the amino acid sequence R(Q)K(R)RAA (i.e. the amino acids Arginine, (Glutamine), Lysine, (Arginine), Arginine, Alanine, Alanine) at position 70-74 in the third hypervariable region are belonging to the Shared Epitope (SE) alleles. This epitope is present in the HLA-DRB1*0101, *0102, *0401, *0404, *0405, *0408, *0410, *1001 and *1402 alleles. The SE is associated with an increased risk (about 2.5 times (33)) to develop RA and a more severe disease course. It is postulated that this SE sequence, which is present in the binding cleft of the HLA-DR molecule, is directly related to the binding of RA inducing peptides to the SE-containing HLA-DR molecules. These peptides are then presented to T cells thought to play an important role in the pathogenesis of RA. Since there is high linkage disequilibrium between HLA-DRB1 and HLA-DQB1 alleles, it is also hypothesized that the HLA-DQB1 alleles are associated with RA susceptibility, although these associations cannot be distinguished (34-36). RA-inducing peptides are not identified yet, but several findings indicate new directions for epitope discovery. Recently it has been shown that SE-containing HLA-DRB1 alleles do not confer risk to the development of RA itself, but predispose to the development of anti-citrullinated protein antibodies (ACPA). These antibodies are highly predictive for RA development as discussed in a previous section (6;7). ACPA+ UA Patients have approximately

Figure 2. HLA class II molecule (adapted from Boots et al. (32)). The variable regions of the - and -chain build up the peptide binding groove. The rectangle indicated in the figure shows the position of the third hypervariable region (HVR) where the shared epitope (SE) or “DERAA”-sequence can be present.

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fifteen times higher odds to develop RA within one year compared to ACPA- UA patients. These ACPA are commonly measured in the IgG isotype, but are also present in the IgM and IgA isotype (37).

The presence of IgG ACPA indicates the presence of T cell help. One of the proteins described to be citrullinated in vivo and present in the synovial fluid of RA patients is the cytoskeletal protein vimentin (38;39). We have recently shown that 90% of ACPA+ RA patients recognizing a citrullinated peptide derived from vimentin carry one or two SE-containing HLA-DRB1 alleles (40), suggesting the involvement of helper T cells recognizing a citrullinated epitope from vimentin in the context of the SE-containing HLA-DRB1 alleles. The identification of citrullinated vimentin-derived T cell epitopes recognized by HLA-DR4 positive individuals is described in Chapter 4 of this thesis.

“DERAA”

“DERAA” stands for the amino acid sequence of Aspartic Glutamic acid-Arginine- Alanine-Alanine which is present in the HLA-DRB1 alleles of the subtype HLA-DRB1*0103, *0402, *1102, *1103, *1301, *1302 and *1304. These alleles are present in 29% of the population (33;41-43).It has been shown by several groups that the frequency of “DERAA”-containing HLA-DRB1 alleles is reduced in RA patients compared to healthy controls and that the risk to develop RA is reduced by about 40% in DERAA positive individuals (33). Since the “DERAA” sequence is positioned at the same location as the SE in the HLA-DRB1 molecule, the effect of “DERAA” has to be evaluated after stratification for presence of the SE-containing HLA-DRB1 alleles. In this way, it has been shown that the effect of “DERAA”-containing HLA-DRB1 alleles is independent of the SE-containing HLA-DRB1 alleles (33).

“DERAA”-containing HLA-DRB1 alleles can be inherited, but can also be conferred as non-inherited maternal antigens (NIMA). NIMA can be conferred from the mother during and/or shortly after the pregnancy since the immune systems of mother and child are in close contact and cell trafficking will occur (44-47). The phenomenon of NIMA was described for the first time in 1954 for the RhD antigen (48) and is illustrated in Figure 3. The terminology is oriented from the point of view of the child in a family, since most studies are coming from the transplantation field. It has been described that haplo-identical NIMA-mismatched sibling transplants have a graft survival similar to that of HLA-identical siblings in contrast to NIPA-mismatched siblings, indicating tolerance for the HLA-mismatch from the mother (49-51).

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The phenomenon of protection against RA by “DERAA”-containing HLA-DRB1 alleles as NIMA is studied in Chapter 2 of this thesis and discussed in comparison with the inherited effect in Chapter 3.

Next to HLA-DRB1 there are several other genes involved in the risk for RA development. Below, only the additional risk alleles studied in the context of this thesis are discussed.

PTPN22

The protein Tyrosine phosphatase named Lyp is encoded by the protein Tyrosine phosphatase, non-receptor type 22 (PTPN22) gene and is expressed by many cell types present in haematopoietic tissues, like T cells, B cells, NK cells, monocytes, dendritic cells and neutrophils (52).

Genes can differ in their nucleotide sequence between different individuals in a population. When the frequency of a single nucleotide change is equal or higher than 1%, this is called a single nucleotide polymorphism (SNP). The most studied SNP of the PTPN22 gene, the C1858T missense single-nucleotide polymorphism, is associated with RA, UA (52-56) and several other autoimmune diseases (57-59). Upto now, it seems that the SNP does not have an effect on the severity of RA, only on the susceptibility (53;60).

There is a large variation in allele frequency among different ethnic populations among the world of the T-variant of the allele, with a variation only in Europe from

2-Figure 3. Terminology of non-inherited maternal antigen (NIMA) and non-inherited paternal antigen (NIPA). The terminology is orientated from the point of view of the child.  gender can be male or female.

NIMA:

NIPA:

CD

AD

AB

B D CB AC

B C A C A D B D NIMA: NIPA: CD AD AB

B D CB AC

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3% in Italians to 15% in Finnish people (61). There are several articles describing the association of the PTPN22 T allele of the C1858T polymorphism with the development of ACPA+ RA, therefore implicating a correlation of this SNP with the production of ACPA (62-64).

We investigated in Chapter 5 whether the C1858T polymorphism of the PTPN22 gene can give additive value to the prediction of progression from UA to RA when it is combined with presence of ACPA.

Genome wide association studies (GWAS)

In the past few years several genome wide association studies (GWAS) have been performed to scan the entire genome for common polymorphisms associating with different autoimmune diseases, including RA (65-68). Since thousands of patients and controls from different populations are studied in the GWAS, these studies may identify risk factors with modest effects on the risk for RA development, and also allow one to study subpopulations of patients for specific effects.

From all genes identified to be associated with RA, we studied based on a recent GWAS (67) six newly identified SNPs for their influence on the severity of RA (Chapter 6). These SNPs are located around genes that are either involved in activation of the immune response (CD40, TNFRSF14, CCL21 and PRKCQ) or play a role in intracellular processes involving cell cycle and homeostatis (MMEL1, KIF5A and CDK6).

Statistical modelling

Several statistical models may be applied to the analysis of genetic associations. The kind of statistical model appropriate for the analysis is dependent on the correlation between the variables and measurement groups, the distribution of the data and the type of study performed. Below, two models are discussed that are used for the different genetic association chapters described in this thesis.

Theory of Bayes

To study the effect of numerous factors on the development of a disease, modelling of the risk factors studied is performed to fit the observed data with the expected frequencies. Modelling of all these risk factors preferably results in prediction of the outcome. A theory that calculates the probability that a hypothesis is true based on the available information is the theory of Bayes, which is used for the studies performed in

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Chapter 2 and 5 of this thesis. With this theory, a prior probability (e.g. to develop RA), based on the risk of an individual in a certain population, is converted into a posterior probability, calculated on the basis of extra information derived from the risk factors studied. The prior probability is most often the prevalence of e.g. a certain disease (or symptom) in the population (69-71).

Linear Mixed Model

A mixed model is a multiple variance analysis often used to compare groups of individuals including multiple measurements from one individual e.g. in time. The data of every individual are plotted in a linear way, and combined for the whole group, thereby intrapolating missing values (72). In a mixed model both random and fixed effects are included. Fixed effects are categorical variables from which all levels are fixed and known, whereas random effects are variables where only a random sample of possible values is measured. A Gaussian distribution is assumed for the variables studied. Since all individual measurements are taken into account in one analysis, it is a powerful method with low intra-individual variability and therefore smaller group sizes are required (73). A linear mixed model was used in Chapter 6 of this thesis to study the effect of different SNPs on the severity of RA.

Outline of the thesis

Chapter 2 reports a family study in which we studied whether “DERAA”-containing HLA-DRB1 alleles protect against the development of rheumatoid arthritis not only when the alleles are inherited but also when they are present as a non-inherited maternal antigen (NIMA).

Chapter 3 is a review that summarizes the epidemiological findings about “DERAA”-containing HLA-DRB1 alleles and RA and the observation we made in Chapter 2. Both associations are compared and possible explanations for the protective phenomenon are described.

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could identify possible T cell epitopes from an important ACPA target antigen, namely the citrullinated vimentin protein.

Both for ACPA and the C1858T polymorphism of the PTPN22 gene associations with RA development have been described. We studied the individual contribution of ACPA and a PTPN22 SNP on the prediction of RA development from UA in Chapter 5. We also studied the influence of the PTPN22 SNP on the level of ACPA.

Genes can be involved both in the susceptibility and severity of RA. This is the case e.g. for the SE-containing HLA-DRB1 alleles. Therefore, we studied in Chapter 6 the contribution of several SNPs that were newly identified as risk factors for the development of RA and the severity of the disease.

The results obtained in the chapters 2-6 of this thesis are summarized and discussed in Chapter 7.

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(52) Begovich AB, Carlton VE, Honigberg LA, Schrodi SJ, Chokkalingam AP, Alexander HC et al. A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 2004; 75(2):330-337. (53) Wesoly J, van der Helm-van Mil AH, Toes RE, Chokkalingam AP, Carlton VE, Begovich AB

et al. Association of the PTPN22 C1858T single-nucleotide polymorphism with rheumatoid arthritis phenotypes in an inception cohort. Arthritis Rheum 2005; 52(9):2948-2950. (54) Seldin MF, Shigeta R, Laiho K, Li H, Saila H, Savolainen A et al. Finnish case-control and

family studies support PTPN22 R620W polymorphism as a risk factor in rheumatoid arthritis, but suggest only minimal or no effect in juvenile idiopathic arthritis. Genes Immun 2005; 6(8):720-722.

(55) Hinks A, Barton A, John S, Bruce I, Hawkins C, Griffiths CE et al. Association between the PTPN22 gene and rheumatoid arthritis and juvenile idiopathic arthritis in a UK population: further support that PTPN22 is an autoimmunity gene. Arthritis Rheum 2005; 52(6):1694-1699. (56) Dieude P, Garnier S, Michou L, Petit-Teixeira E, Glikmans E, Pierlot C et al. Rheumatoid

arthritis seropositive for the rheumatoid factor is linked to the protein tyrosine phosphatase nonreceptor 22-620W allele. Arthritis Res Ther 2005; 7(6):R1200-R1207.

(57) Reddy MV, Johansson M, Sturfelt G, Jonsen A, Gunnarsson I, Svenungsson E et al. The R620W C/T polymorphism of the gene PTPN22 is associated with SLE independently of the association of PDCD1. Genes Immun 2005; 6(8):658-662.

(58) Kahles H, Ramos-Lopez E, Lange B, Zwermann O, Reincke M, Badenhoop K. Sex-specific association of PTPN22 1858T with type 1 diabetes but not with Hashimoto's thyroiditis or Addison's disease in the German population. Eur J Endocrinol 2005; 153(6):895-899.

(59) Skorka A, Bednarczuk T, Bar-Andziak E, Nauman J, Ploski R. Lymphoid tyrosine phosphatase (PTPN22/LYP) variant and Graves' disease in a Polish population: association and gene dose-dependent correlation with age of onset. Clin Endocrinol (Oxf) 2005; 62(6):679-682. (60) Naseem H, Thomson W, Silman A, Worthington J, Symmons D, Barton A. The

PTPN22*C1858T functional polymorphism is associated with susceptibility to inflammatory polyarthritis but neither this nor other variants spanning the gene is associated with disease outcome. Ann Rheum Dis 2008; 67(2):251-255.

(61) Gregersen PK, Lee HS, Batliwalla F, Begovich AB. PTPN22: setting thresholds for autoimmunity. Semin Immunol 2006; 18(4):214-223.

(62) Orozco G, Pascual-Salcedo D, Lopez-Nevot MA, Cobo T, Cabezon A, Martin-Mola E et al. Autoantibodies, HLA and PTPN22: susceptibility markers for rheumatoid arthritis. Rheumatology (Oxford) 2008; 47(2):138-141.

(63) Kokkonen H, Johansson M, Innala L, Jidell E, Rantapaa-Dahlqvist S. The PTPN22 1858C/T polymorphism is associated with anti-cyclic citrullinated peptide antibody-positive early rheumatoid arthritis in northern Sweden. Arthritis Res Ther 2007; 9(3):R56.

(64) Kallberg H, Padyukov L, Plenge RM, Ronnelid J, Gregersen PK, van der Helm-van Mil AH et al. Gene-gene and gene-environment interactions involving HLA-DRB1, PTPN22, and smoking in two subsets of rheumatoid arthritis. Am J Hum Genet 2007; 80(5):867-875. (65) Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of

seven common diseases and 3,000 shared controls. Nature 2007; 447(7145):661-678. (66) Fung EY, Smyth DJ, Howson JM, Cooper JD, Walker NM, Stevens H et al. Analysis of 17

autoimmune disease-associated variants in type 1 diabetes identifies 6q23/TNFAIP3 as a susceptibility locus. Genes Immun 2009; 10(2):188-191.

(67) Raychaudhuri S, Remmers EF, Lee AT, Hackett R, Guiducci C, Burtt NP et al. Common variants at CD40 and other loci confer risk of rheumatoid arthritis. Nat Genet 2008; 40(10):1216-1223.

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(68) Barton A, Thomson W, Ke X, Eyre S, Hinks A, Bowes J et al. Rheumatoid arthritis susceptibility loci at chromosomes 10p15, 12q13 and 22q13. Nat Genet 2008; 40(10):1156-1159.

(69) Freedman L. Bayesian statistical methods. A natural way to assess clinical evidence. British medical journal 1996; 313:569-570.

(70) Petrie A, Sabin C. Medical Statistics at a glance. 109-110. 2000. Ref Type: Serial (Book,Monograph)

(71) Vandenbroucke JP, Hofman. Diagnostiek van ziekte. Grondslagen der Epidemiologie. 6th ed. 2004.

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Chapter 2

Protective effect of non-inherited maternal HLA-DR

antigens on Rheumatoid Arthritis development

Anouk L. Feitsma1,2, Jane Worthington3, Annette H.M. van der Helm-van Mil2, Darren Plant3, Wendy Thomson3, Jennie Ursum4, Dirkjan van

Schaardenburg4, Irene E. van der Horst-Bruinsma5, Jon J. van Rood1, Tom W.J. Huizinga2, René E.M. Toes2, René R.P. de Vries1

1. Department of Immunohematology and Blood Transfusion, LUMC, Leiden, The Netherlands

2. Department of Rheumatology, LUMC, Leiden, The Netherlands 3. Arthritis Research Campaign Epidemiology Unit, The University of

Manchester, Manchester, United Kingdom

4. Jan van Breemen Institute, Amsterdam, The Netherlands

5. Department of Rheumatology, VUMC, Amsterdam, The Netherlands

PNAS 2007;104(50):19966-19970

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Abstract

Rheumatoid arthritis (RA) is a complex genetic disorder in which the HLA-region contributes most to the genetic risk. HLA-DRB1-molecules containing the amino-acid sequence “DERAA” (i.e. HLA-DRB1*0103, *0402, *1102, *1103, *1301, *1302 and *1304) are associated with protection from RA. It has been proposed that not only inherited but also non-inherited HLA-antigens from the mother (NIMA) can influence RA-susceptibility. Up to now, no protective NIMAs were described. Here, we studied whether “DERAA”-containing HLA-DRB1-alleles as NIMA are associated with a protective effect.

Hundred-seventy-nine families were studied, 88 from the Netherlands and 91 from the UK. The frequency of “DERAA”-containing HLA-DRB1-alleles of the Dutch mothers (16.1%), but not of the fathers (26.2%), was lower compared to the general Dutch population (29.3%; p=0.02). This was replicated in the English set of patients and controls (p=0.01). Further, of all families, 45 contained at least one “DERAA”-negative child with RA and at least one “DERAA”-positive parent. The odds for the “DERAA”-negative RA patients of having a “DERAA”-positive mother was significantly lower as compared to having a “DERAA”-positive father (OR 0.25; p=0.003).

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Introduction

Rheumatoid arthritis (RA) is a complex genetic disorder in which the HLA-region contributes most to the genetic risk. Especially HLA-DRB1 molecules sharing a common epitope, R(Q)K(R)RAA,(i.e. the amino acids Arginine, (Glutamine), Lysine, (Arginine), Arginine, Alanine, Alanine) at position 70-74, the so-called shared epitope (SE), are associated with both susceptibility to and severity of RA (1-4). At the same position of the HLA-DRB1 molecules as the SE, the amino acids “DERAA” (i.e. the amino acids Aspartic acid, Glutamic acid, Arginine, Alanine, Alanine) can be present. Individuals carrying HLA-DRB1 alleles that express this “DERAA”-sequence (“DERAA”-positive individuals) (“DERAA” is present in HLA-DRB1*0103, *0402, *1102, *1103, *1301, *1302 and *1304) have a lower susceptibility to develop RA and less severe disease compared to individuals with ‘neutral’ (SE- and “DERAA”-negative) HLA-DRB1 alleles. “DERAA”-containing HLA-DRB1 alleles protect in both SE-negative and SE-positive individuals and therefore this effect is independent of the effect of SE-alleles (5).

It has been proposed that not only inherited but also non-inherited HLA-antigens from the mother (NIMA) as opposed to those from the father (NIPA) can influence the immune reactivity of an individual with implications for tissue transplant survival and susceptibility to autoimmune disease (6-8). During pregnancy the immune systems of mother and child are in close contact and trafficking of cells, antibodies and/or antigens can occur. Confrontation of the fetal/newborn immune system with the NIMA may have a lifelong influence on the immune response of the child. It has been shown in transplantation studies, that haplo-identical NIMA-mismatched sibling transplants had a graft survival similar to that of HLA-identical siblings, whereas NIPA-mismatched sibling transplants did as poorly as did recipients of maternal and paternal grafts (9). We have described that HLA-DR4 or SE NIMA but not HLA-DR4 or SE NIPA are associated with susceptibility to RA, because HLA-DR4 or SE-negative RA patients have more often a HLA-DR4 or positive mother compared to a HLA-DR4 or SE-positive father (10, 11). This observation was confirmed in one study (8) while in two other studies there was a non-significant trend in the same direction (12, 13). When the studies were combined a significant HLA-DR4 and SE NIMA effect in DR4 or SE negative patients was observed (8). This is not or less clearly the case for HLA-DR4 or SE-positive RA patients (10, 11, 14). The strongest genetic risk factors for type I

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diabetes, HLA-DR3-DQ2 and HLA-DR4-DQ8, are also more frequent in mothers as compared to fathers of patients negative for one or both of these antigens (7).

As there is so far no evidence for a protective effect in human autoimmune disease for NIMAwe were interested to study whether “DERAA”-containing HLA-DRB1 alleles as NIMA are associated with a protection against RA.

To answer this question, 88 Dutch and 91 English families were typed for HLA-DRB1. Families in which the RA patient did not carry a HLA-DRB1 allele containing “DERAA” and either the father, the mother or both carried “DERAA”-containing HLA-DRB1 alleles, were analyzed for the presence of a NIMA effect mediated by “DERAA”-containing HLA-DRB1 loci.

Patients and Methods

Dutch RA families: 88 consecutive patients with RA fulfilling the 1987 ACR criteria were recruited in 1996 in two outpatient clinics: 37 from the Leiden University Medical Centre, Leiden, and 51 from the Jan van Breemen institute, Amsterdam. At time of inclusion, both parents of the patient had to be alive. Blood samples were drawn from patients and their parents to perform HLA-DRB1 typing.

Dutch Controls: A randomly selected panel of 423 healthy unrelated Dutch indivi-duals served as control population for the Dutch HLA-DRB1 allele frequencies (5).

Dutch control families: HLA-DRB1 typings of 208 healthy mothers and child pairs were analyzed to control for the specificity of a possible NIMA effect of “DERAA”-containing HLA-DRB1 alleles in the RA families. These families were collected from a database (36) that includes deliveries that took place in of the Obstetric Department of the Leiden University Medical Centre.

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English controls: An English Caucasian study population from the Allele Frequency Database consisting of 177 individuals was used as control population for the English HLA-DRB1 allele frequencies (39).

HLA genotyping

HLA-DRB1 alleles were determined in all RA patients, their parents, brothers, sisters and controls. In the English families, seven typings of the HLA-DRB1 alleles of either the mother or the father were deduced from the alleles present in the other family members.

HLA-DRB1 typing for the Dutch individuals was performed as described previously (11). In England HLA-DRB1 typing was performed by polymerase chain reaction, using specific primers and hybridization with sequence-specific biotin labeled oligonucleotides (Dynal kit, Dynal Biotech, Wirral, UK). In four of the 88 fathers and one of the 88 mothers no definitive HLA-DRB1 allele could be assigned. Therefore, these individuals were excluded from the analysis.

The following HLA-DRB1 alleles were classified as containing the “DERAA” epitope: HLA-DRB1*0103, *0402, *1102, *1103, *1301, *1302 and *1304.

Statistics

The patient characteristics of the Dutch and English patients were compared with either a Chi-square (dichotomous variables) or independent T-test (continuous variables). For the patient groups of table 3 (<30 individuals per group), the patient characteristics were compared with the Fischer exact and Mann-Whitney tests.

The “DERAA” frequencies of the mothers and the fathers of both the Dutch and English RA patients were compared separately to the “DERAA” frequency in the Dutch and English healthy control populations, respectively, by using a Chi-square test. In the Dutch healthy control population, the frequency of “DERAA”-containing HLA-DRB1 alleles in women and men was also compared.

An association between the presence and absence of the “DERAA”-containing HLA-DRB1 alleles as a NIMA or a NIPA was calculated using odds ratios with 95% confidence intervals combined with a Chi-square test. The observed frequency of “DERAA”-positive mothers was compared to the expected frequency using a binomial test. The expected frequency was calculated with the method of Bayes and a comparable distribution of the English and Dutch families contributing to this analysis was taken into account for the calculation of the expected frequency. These analyses

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were performed for parents of patients not carrying a “DERAA”-containing HLA-DRB1 allele.

The Chi-square, independent T-tests and the Binomial test were performed using SPSS_12.0 Software (Chicago, IL, USA). The odds ratios and 95% confidence inter-vals were calculated using Statcalc Software (EpiInfo version 5, Statcalc, December 1990).

Results

Two different data sets were studied: Dutch RA patients with their parents and English RA patients with their brothers, sisters and parents. The characteristics of both data sets at the time of taking the blood sample for HLA-DRB1 typing are listed in Table 1.

Table 1. Clinical and laboratory characteristics of the Dutch and English patients used for this study.

Dutch English

(n=88) (n=223*)

Age at onset (years) 30 32

Disease duration (years) 7.8 11.5

female sex (%) 86.5 79.8

Rheumatoid Factor positive (%) 57 84

SE positive (%) 74 86

Erosive disease (%) 87 84

The age at onset and disease duration show the mean values in years. Disease duration is the duration of rheumatoid arthritis at the time of taking the sample for HLA-DRB1 typing. The positivity of rheumatoid factor was also determined at the time of taking the blood sample for HLA-DRB1 typing. *out of 91 (multi-case) families.

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multi-case families more often are carriers of predisposing HLA-DRB1 alleles (the SE- alleles), often have more severe disease and therefore have a higher frequency of rheumatoid factor antibodies (15). Since these differences were as expected and were not considered to interfere with our research question, the patients from both data sets were pooled for some analyses.

The frequency of “DERAA”-containing HLA-DRB1 alleles present in the Dutch RA patients (“DERAA”-positive RA patients) (14.6%) was significantly lower than that of the Dutch healthy control population (29.3%; p=0.007). A similar observation was made in the English patients (only the oldest RA child of every family was included) as the frequency of “DERAA”-containing HLA-DRB1 alleles (8.6%) was significantly lower than that of the English control population (23.8%; p=0.002). These data confirm the protective effect associated with “DERAA”-containing HLA-DRB1 alleles. Before studying a possible effect of “DERAA”-containing HLA-DRB1 alleles as NIMA, we studied whether there was no difference in inheritance of “DERAA”-containing HLA-DRB1 alleles from fathers or mothers to their children. Therefore, we analyzed the frequency of fathers and mothers that have passed on a “DERAA”-containing HLA-DRB1 allele to “DERAA”-positive RA patients. As expected, “DERAA”-containing HLA-DRB1 alleles were equally inherited from fathers or mothers in both the Dutch and English families (data not shown). These data indicate that there is no gender difference in inheritance of “DERAA”-containing HLA-DRB1 alleles.

If non-inherited “DERAA”-containing HLA-DRB1 alleles of the mother protect the child to RA development, it is expected that the frequency of mothers of RA patients bearing a “DERAA”-containing HLA-DRB1 allele is lower compared to the general population. Therefore, we determined whether the frequency of “DERAA”-containing HLA-DRB1 alleles of mothers and fathers of RA patients was different as compared to controls. The frequencies of “DERAA”-containing HLA-DRB1 alleles of the mothers and fathers of the 88 Dutch RA families were therefore compared with the frequency of “DERAA”-containing HLA-DRB1 alleles of a Dutch healthy control population (Table 2). Twenty-two Dutch fathers (26.2%) carried a “DERAA”-containing HLA-DRB1 allele whereas in only 14 mothers (16.1%) a “DERAA”-containing HLA-allele was present. When these frequencies were compared to the frequency of “DERAA”-containing HLA-DRB1 alleles in a Dutch healthy control population (29.3 %), the mothers showed a significantly lower frequency (p=0.02) compared to the control

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population. In contrast, the frequencies of the fathers of the RA patients and the individuals of the healthy control group were comparable.

Table 2 “DERAA” frequency of mothers and fathers of Dutch and English RA patients compared with healthy Dutch and English controls.

“DERAA”+ “DERAA”- frequency OR (95% CI) p-value

n = n = (%)

Dutch

Mothers of RA patients 14 73 16.1 0.46 (0.24-0.88) 0.02*

Fathers of RA patients 22 62 26.2 0.86 (0.49-1.50) 0.66

Contr. Fam. Mothers 67 141 32.2 1.15 (0.79-1.67) 0.51

Healthy controls 124 299 29.3

English

Mothers of RA patients 9 82 9.9 0.35 (0.15-0.80) 0.01*

Fathers of RA patients 14 75 15.7 0.60 (0.29-1.22) 0.18

Healthy controls 42 135 23.8

“DERAA”+: carriership of one or two “DERAA” containing HLA-DRB1 alleles. “DERAA”-: no “DERAA”- containing HLA-DRB1 allele present. Contr. Fam. Mothers: Mothers of the control population from the Department of Obstetrics of the Leiden University Medical Centre. OR= odds ratio compared to healthy controls. 95% CI= 95% confidence interval.

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To exclude the possibility that the difference in “DERAA”-containing HLA-DRB1 allele frequency between the mothers and fathers is due to a general difference in “DERAA”-containing HLA-DRB1 allele frequency between males and females, the frequencies of “DERAA”-containing HLA-DRB1 alleles in males and females of the Dutch healthy control cohort were analyzed. Fifty out of 186 women carried one or two “DERAA”-containing HLA-DRB1 alleles (26.8%) compared to 67 out of 232 men (29.5%). These frequencies were not significantly different (OR= 0.91; 95%CI 0.58-1.42; p=0.73), indicating that the lower frequency of “DERAA”-containing HLA-DRB1 alleles in the mothers as compared to the fathers of RA patients points to a mother-specific effect of “DERAA”-containing HLA-DRB1 alleles on the child.

To further ascertain that the observed difference in frequency of “DERAA”-containing HLA-DRB1 alleles between mothers and fathers of RA patients could indeed be attributed to an effect of non-inherited HLA-antigens, the “DERAA”-positive families with a “DERAA”-negative child (the RA patient) were selected for further analysis. The patient characteristics of this group were comparable to the data shown in Table 1 except for a borderline significant difference in sex in the English patient group (p=0.04). Since the patient characteristics between the Dutch and English patients (as shown in Table 1) only differed for the expected characteristics (RF, SE and disease duration), the patients were pooled for further analysis. From the 45 families fulfilling the selection criterion, 17 “DERAA”-positive mothers and 32 “DERAA”-positive fathers were identified (Table 3).

Table 3 Mothers of negative RA patients carry less often a “DERAA”-containing HLA-DRB1 allele than fathers.

DERAA+ DERAA- frequency OR (95% CI) p-value

n = n = (%)

Mothers 17 28 37.8 0.25 (0.09-0.65) 0.003*

Fathers 32 13 71.1

The data of the English and Dutch families are combined.

“DERAA”+: carriership of at least one “DERAA”-containing HLA-DRB1 allele. “DERAA”-: no “DERAA”- containing HLA-DRB1 allele present. The frequency is the percentage DERAA-positive individuals. OR= odds ratio of mothers compared to fathers. 95% CI= 95% confidence interval.

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The odds ratio (OR) for negative RA patients of having a “DERAA”-positive mother compared to a “DERAA”-“DERAA”-positive father was 0.25 (95% CI 0.09-0.65; p=0.003). The observed frequency of “DERAA”-positive mothers (37.8%) was also significantly decreased compared to the expected frequency (53.6%; p=0.02). When the data of the 45 families were stratified for SE status of the patient (i.e. either no SE alleles or heterozygous or homozygous for SE) no significant differences were observed between the OR of the DERAA-NIMA versus -NIPA between the different subgroups (data not shown), indicating that the observed NIMA effect of DERAA-containing HLA-DRB1 alleles is probably independent of SE status. However the numbers in the different subgroups were small, particularly for the SE negative patients.

Finally to exclude that also in non-RA families there is a NIMA effect of “DERAA”-containing HLA-DRB1 alleles, a Dutch control population (mother-child pairs from the LUMC Department of Obstetrics) was analyzed. The frequency of “DERAA”-containing HLA-DRB1 alleles in both the mothers (32.2%, Table 2) and children (30.3%) were comparable with that of the Dutch healthy control population (29.3%), showing that there is no (NIMA) effect of “DERAA”- containing HLA-DRB1 alleles in healthy control families. These results together show that there is a protective effect of “DERAA”-containing HLA-DRB1 alleles as NIMA on development of RA of the child.

Discussion

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“DERAA”-positive father was 0.25. These data show a protective NIMA-effect in a human autoimmune disease and indicate that a “DERAA”-positive mother can transfer protection against RA to her “DERAA”-negative child.

HLA-DRB1 molecules play a large role in the genetic risk of developing RA. At position 70-74 of the HLA-DRB1 molecules either the amino acids of the SE (R(Q)K(R)RAA) can be present or the amino acids “DERAA”. The odds ratio of individuals carrying HLA-DRB1 alleles that express the “DERAA”-sequence (HLA-DRB1*0103, *0402, *1102, *1103, *1301, *1302 and *1304) compared to individuals with “neutral” (SE- and “DERAA”-negative) HLA-DRB1 alleles to develop RA is 0.5-0.7, indicating that “DERAA”-positive individuals have a lower susceptibility to develop RA (5, 19-21). Since the odds ratio of “DERAA” was corrected for SE-alleles, it can be concluded that the “DERAA”-containing HLA-DRB1 alleles are independently associated with a reduced risk to develop RA. The mechanism of protection is unknown, but it has been proposed that it is mediated by T cells recognizing peptides containing the “DERAA”-sequence presented by HLA-DQ molecules (22). Whether these T cells have a regulatory phenotype or are deleted in the thymus by negative selection is still a subject of research. Our observation of a protective effect of “DERAA”-containing HLA-DRB1 alleles as NIMA on RA development gives a new dimension to the direction of this research.

During pregnancy, cells of the mother migrate to the fetus and may induce lifelong microchimerism in the child (23-25). Maternal microchimerism has been shown in mice to induce neonatal B cell (26) and probably also T cell (27) tolerance and is therefore one of the possible mechanisms for NIMA effects (28). Although speculative, we postulate therefore that the protective effect of the DERAA-containing HLA-DRB1 alleles as NIMA on the development of RA is most probably mediated by maternal cells entering the bloodstream and tissues of the child which exert their effect through a change in the immune repertoire and most likely the T cell repertoire of the child. These maternal cells might influence thymic selection or act in the peripheral lymphoid organs, for example as a consequence of the sustained presence of cells from the mother in the child. It has been shown that maternal microchimeric cells can be present in many different cell subsets (29) in both healthy and diseased individuals (30, 31) in which they may exert different effects (32, 33). Likewise, immune regulatory mechanisms might directly be induced in the fetus as it has recently been described that the fetus can already develop cytotoxic T cells directed at a maternal minor H antigen

(36)

in utero (34) or becomes sensitized against foreign antigens to which the mother is exposed during pregnancy (35).

Further studies on the intriguing interplay between the developing immune system of the child and cells from the mother are needed both to increase our understanding on how NIMA can influence the immune system of the child and to learn whether and if so how this might be used to combat autoimmune diseases.

Acknowledgements

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Chapter 3

Protection against Rheumatoid Arthritis by HLA:

Nature and Nurture

Anouk L. Feitsma1,2, Annette H.M. van der Helm-van Mil1, Tom W.J. Huizinga1, René R.P. de Vries2, René E.M. Toes1

1. Department of Rheumatology, LUMC, Leiden, The Netherlands 2. Department of Immunohematology and Blood Transfusion, LUMC,

Leiden, The Netherlands

Ann Rheum Dis 2008;67:iii61-iii63

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Abstract

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Summary

Rheumatoid arthritis (RA) is a complex genetic disorder in which the HLA-region contributes most to the genetic risk. HLA-DRB1-molecules containing the amino-acid sequence QKRAA/QRRAA/RRRAA (i.e. HLA-DRB1*0101, *0102, *0401, *0404, *0405, *0408, *0410, *1001 and *1402) at position 70 to 74 in the third hypervariable region of the DRB1 chain are associated with susceptibility to RA. HLA-DRB1 molecules containing the amino acids “DERAA” (i.e. HLA-DRB1*0103, *0402, *1102, *1103, *1301, *1302 and *1304) at the same position are associated with protection from RA.

Interestingly, not only inherited but also non-inherited HLA-antigens from the mother can influence RA-susceptibility. We have recently described a protective effect of “DERAA”-containing HLA-DRB1 alleles as non-inherited maternal antigen (NIMA). The underlying mechanism of this protective effect is currently unknown, although a possible explanation is covered below. In this review, an overview of the current knowledge on protection against RA is given and the inherited and NIMA effect of “DERAA”-containing HLA-DRB1 alleles are compared.

HLA-DRB1 "DERAA"-positive alleles protect against RA

Rheumatoid arthritis (RA) is a complex genetic disorder in which the HLA-region contributes most to the genetic risk. Especially HLA-DRB1 molecules sharing a common epitope, R(Q)K(R)RAA, (i.e. the amino acids Arginine, (Glutamine), Lysine, (Arginine), Arginine, Alanine, Alanine) at position 70-74 in the third hypervariable region of the DRB1 chain, the so-called shared epitope (SE), are associated with both susceptibility to and severity of RA (1-4). The shared epitope is present in the HLA-DRB1*0101, *0102, *0401, *0404, *0405, *0408, *0410, *1001 and *1402 molecules. At the same position as the SE, the amino acids “DERAA” (i.e. the amino acids Aspartic acid, Glutamic acid, Arginine, Alanine, Alanine) can be present in other HLA-DRB1 molecules (i.e. HLA-HLA-DRB1*0103, *0402, *1102, *1103, *1301, *1302 and *1304). Individuals carrying HLA-DRB1 alleles that express this “DERAA”-sequence display a lower susceptibility to develop RA and suffer from less severe disease as compared to individuals with ‘neutral’ (SE- and “DERAA”-negative) HLA-DRB1 alleles. The odds ratio of individuals carrying HLA-DRB1 alleles that express the sequence compared to individuals with “neutral” (SE- and negative) HLA-DRB1 alleles to develop RA is 0.5-0.7, indicating that

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positive individuals have a lower susceptibility to develop RA (5-8). The protective effect associated with “DERAA” is also found after stratification for the presence or absence of HLA-SE alleles. This indicates that the protective effect associated with “DERAA”-expression cannot be explained by an overrepresenation of SE alleles in patients, resulting automatically in a lower frequency of other HLA alleles in RA patients. Thus, the “DERAA”-containing HLA-DRB1 alleles are independently associated with a reduced risk to develop RA (5).

It is unclear whether the entire “DERAA” motif is essential for the protection or that only certain amino acids of this motif confer the same effect. In contrast to several reports showing the protective effects by “DERAA”-containing HLA-DRB1 alleles to the development and severity of RA (5,9,10), other reports hypothesize that the amino acids “RAA” at position 72-74 in the third hypervariable region influence the susceptibility to RA development whereas the amino acids at position 70 and 71 modulate this effect (11,12). In these articles it is indicated that HLA alleles expressing the 70ERAA74 sequence or the Aspartic acid (D) at position 70 both have a lower frequency in RA patients as compared to healthy controls. Further, it has also been described that protection is mainly associated with the Aspartic acid (D) at position 70 (8,13).

Thus, despite these differences in nomenclature and stratification, it is getting increasingly clear that some HLA alleles confer susceptibility, whereas others are associated with protection.

The mechanism of protection is unknown, but it has been proposed that it is mediated by T cells recognizing peptides containing the “DERAA”-sequence presented by HLA-DQ molecules (14). Whether these T cells have a regulatory phenotype or are deleted in the thymus by negative selection is still a subject of research.

Non-inherited "DERAA" from the mother also

gives protection to the child for RA development

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NIMA are coming from the transplantation field. Claas et al. described that renal transplant patients often do not generate antibodies against the mismatched HLA antigens of their mother in comparison to those of their father and are therefore tolerant for this HLA mismatch when they are transplanted. This is associated with a longer transplant survival (16-18). This is exemplified best in a study by Burlingham showing that haplo-identical NIMA-mismatched sibling transplants have a graft survival similar to that of HLA-identical siblings, whereas NIPA-mismatched sibling transplants did as poorly as did recipients of maternal and paternal grafts (19).

We have recently shown that there is also a protective effect on the development of RA of HLA-DRB1 molecules that contain the amino acid sequence “DERAA” when presented as NIMA on the development of RA (20). We anticipated that if non-inherited “DERAA”-containing HLA-DRB1 alleles of the mother protect the child to RA development, it is expected that the frequency of mothers of RA patients bearing a “DERAA”-containing HLA-DRB1 allele is lower compared to the general population. Indeed, using a cohort of Dutch RA patients together with their parents, we were able to show that the mothers of RA patients showed a significantly lower frequency (16.1%) of “DERAA”-containing HLA-DRB1 alleles compared to the Dutch control population (29.3%; p = 0.02). In contrast, the frequencies of “DERAA”-containing HLA-DRB1 alleles in the fathers of the RA patients (26.2%) and the individuals of the healthy control group were comparable. These findings were replicated in the English

Figure 1. Terminology of non-inherited maternal antigen (NIMA) and non-inherited paternal antigen (NIPA). The terminology is orientated from the point of view of the child.  gender can be male or female.

NIMA:

NIPA:

C

D

A

D

A

B

B D

C

B

A

C

B C A C A D B D NIMA: NIPA:

C

D

A

D

A

B

B D

C

B

A

C

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multi-case families from Manchester. To further ascertain that the observed difference in frequency of “DERAA”-containing HLA-DRB1 alleles between mothers and fathers of RA patients could indeed be attributed to an effect of non-inherited HLA-antigens, the “DERAA”-positive families with a “DERAA”-negative child (the RA patient) were selected for further analysis. For this analysis, the patients from the UK and the Netherlands were pooled. The odds ratio (OR) for “DERAA”-negative RA patients of having a “DERAA”-positive mother compared to a “DERAA”-positive father was 0.25 (95% CI 0.09-0.65; p=0.003). These results together show that there is a protective effect of “DERAA”-containing HLA-DRB1 alleles as NIMA on development of RA of the child.

Table 1. Comparison of the inherited and NIMA effect of “DERAA” Mother Child RA patients

(n = 89)

Controls (n = 206)

A pos pos 7 39

B neg pos 6 23

C pos neg 8 26

D neg neg 68 118

The Dutch families were used for this analysis [20]. Group B vs D is the inherited effect. OR = 0.45 (0.16-1.25); Group C vs D reflects the NIMA effect. OR = 0.53 (0.21-1.31). Pos = positive (hetero-zygote) for “DERAA”-containing HLA-DRB1 alleles. Neg = negative for “DERAA”-containing HLA-DRB1 alleles.

Figure

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References

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